ISSN: 0975-8585

Research Journal of Pharmaceutical, Biological and Chemical Sciences Anti-angiogenic and Anti-Inflammatory Activity of Punica granatum Peel on Experimentally -Induced Gastric Ulcer in Rats Roba M Talaat1*, Nehal E Abdel-Hakem1, Sayed A El-Toumy2, Rehab M Samaka3, Azza H Mohamed4, and Mohamed E Ebeed1. 1

Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Egypt. 2 Chemistry of Tannins Department, National Research Center (NRC), Cairo, Egypt. 3 Pathology Department, Faculty of Medicine, Menofiya University, Shebin El-Kom, Egypt. 4 Zoology Department, Faculty of Science, Menofiya University, Shebin El-Kom, Egypt.

ABSTRACT Pomegranate (Punica granatum) has been used in folk medicine for the treatment of gastric ulcer. Thus, the current study aimed to evaluate the reparative and anti-inflammatory activity of methanolic extracts of pomegranate peel (mesocarp and epicarp). This study was conducted on male Wistar rats divided into 9 groups: Group 1; normal controls, Group 2; received 80% ethanol (positive control), Groups 3, 4, 5, 6, 7, 8 and 9; gastric ulcer treated with 250mg/kg, 500mg/kg (mesocarp extract), 250mg/kg, 500mg/kg (epicarp extract), Ranitidine, Omeprazole and Sucralfate, respectively. Serum pro-angiogenic [vascular endothelial and platelet derived growth factors (VEGF and PDGF) and inflammatory mediators [Interleukin (IL-1), IL-6, tumor necrosis factor (TNF-α) and cyclooxygenase (COX-2)] were performed by enzyme-linked immunosorbent assay (ELISA). Pomegranate peel methanolic extracts are found to have antiulcer property. They have been proved as anti-inflammatory agents by reducing gastric mucosal COX-2 and plasma TNF-α level. Healing effect was approved by elevation of PDGF level. Maximum ulcer healing effect was elicited in epicarp extract at 500mg/kg. Taken together, pomegranate peel methanolic extracts could be considered as one of the antiulcer therapeutic strategies based on its anti-inflammatory and reparative activities. Further pharmacokinetic analysis of these extracts will be taken in consideration in future study. Keywords: Punica granatum, Gastric ulcer, Angiogenesis, Inflammation.

*Corresponding author

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ISSN: 0975-8585 INTRODUCTION Peptic ulcer disease (PUD) is the most common gastrointestinal disorder in clinical practice. It is a chronic, multifactorial disease distinguished by different degrees of denudation in gastric or duodenal mucosa ] 1,2[ . Ulcer pathogenesis is resultant of an imbalance between aggressive factors [acid, pepsin, bacterial infection (Helicobacter pylori) and non-steroidal anti-inflammatory drugs (NSAIDs)] and defensive factors (mucus bicarbonate, blood flow, gastric mucin and prostaglandins) that are needed for the maintenance of integrity of gastro-duodenal mucosa ]3[ . Gastric ulcer therapy faces a major drawback in modern days due to the undesirable side effects of the long term uses of commercially available drugs ]4[. The conventional drugs used in the treatment of gastric ulcer include histamine (H2) receptor antagonist, proton pump inhibitors, antacids and anti-chollinergics ]5,6[ . As it affects approximately 15-20% of the global population ]7[ , the treatment of this disease has become one of the challenging problems today. Thus, the search is still on to discover a drug possessing antiulcer activity, which will serve as a powerful therapeutic agent for gastric ulcer. Due to lack of side effects compared to synthetic drugs, new trends in treatment have relied on plants for medication. From ancient times, plants have been proved to be powerful therapeutic agent for the treatment of various human diseases. One of these plants is Punica granatum (Punicaceae), a commonly called pomegranate, is an edible fruit cultivated in Mediterranean countries, Asian countries and some parts of the United States. P. granatum has been widely used by traditional medicine in America, Asia, Africa and Europe for the treatment of different types of diseases ]8,9,10[ . Pomegranate fruit is a rich source of two types of polyphenolic compounds: anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin and ellagic acid esters of glucose). A number of biological activities such as anti-tumour ]11[ , anti-bacterial ]12[ , anti-diarrhoeal ]13[ , and anti-fungal ]14[ activities have been reported with various extracts/constituents of different parts of this plant. Pomegranate is now gaining importance because of its potent antioxidant activity. As some potent antioxidants have been isolated from the fruit juice and have been found to be bioavailable, effective and safe ]15[. Moreover, it has been used previously in folk medicine for the treatment of ulcer ]16[. Inflammation can stimulate angiogenesis, and angiogenesis can facilitate inflammation ]17[. Inflammatory mediators can, either directly or indirectly, promote angiogenesis. Angiogenesis, in turn, contributes to inflammatory pathology. New blood vessels can maintain the chronic inflammatory state by transporting inflammatory cells to the site of inflammation and supplying nutrients and oxygen to the proliferating inflamed tissue. The increased endothelial surface area also creates an enormous capacity for the production of cytokines, adhesion molecules, and other inflammatory stimuli ]18[. Based on these facts, the present work aimed to evaluate in vivo antiulcer, anti-angiogenic and antiinflammatory activity of different methanolic extracts from P. granatum peel (mesocarp and epicarp) and comparing the healing activity of these extracts with three well documented anti-ulcer drugs act by different pharmacokinetics; Omeprazole (Omz) (proton pump inhibitor), Ranitidine (Ran) (histamine H2-receptor antagonist) and Sucralfate (Suc) (cytoprotective agent). MATERIALS AND METHODS Animals Ninty male Wistar rats (body weight 150–200g) were purchased from VACSERA, Egypt. The animals were maintained under standardized environmental conditions (22-28°C, 60-70% relative humidity, 12 h dark/light cycle). They were allowed to access to food and water ad libitum. All experimental protocols described in this study were in accordance with the rules and regulation of the Animal Ethics rules, Menofiya University, Egypt. Plant material The fruits of P. granatum were collected from the local market in Cairo, Egypt in October, 2010 and identified by experts in National Herbarium reference.

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ISSN: 0975-8585 Extraction The peels of P. granatum were manually separated from the whole fruits to epicarp and mesocarp, dried in hot-air woven, powdered and extracted with a mixture of methanol: water (7:3, v/v) by a Soxhlet apparatus at 65 ºC. The solvent was completely removed and the dried crude extract thus obtained was used for investigation. Phytochemical analysis The aqueous methanol extract of the P. granatum epicarp and mesocarp was subjected to qualitative chemical screening for the identification of the tannins, and flavonoids using standard procedures ] 19[. Test for tannins The aqueous extract (1 ml) was mixed with 10 ml of distilled water and filtered. Ferric chloride reagent (3 drops) was added to the filtrate. A blue-black or green precipitate confirmed the presence of gallic tannins or catechol tannins, respectively. Test for flavonoids A portion of the aqueous extract (2 ml) was heated, and metallic magnesium and concentrated hydrochloric acid (5 drops) were added. A red or orange coloration indicated the presence of flavonoids. Ethanol (80%) induction of gastric ulcer in rats and role of P. granatum methanolic extracts The rats were randomly divided into 9 groups (10 animals each). Gastric ulcer was induced as previously described by Paiva et al. ]20[. Group (1) was kept as normal controls without any treatment. All other groups were fasted from 24 to 36h before 80% ethanol administration. All treatments were administered orally, 1h prior to the ethanol administration. All groups were sacrificed 4hr after ethanol administration. Effect of P. granatum extracts were compared with different synthetic drugs such as Ranitidine (MUP, Egypt), Omeprazole (MUP, Egypt) and Sucralfate (EIPICO, Egypt). Group (2) (gastric ulcer, untreated group) was received 1 ml of 80% ethanol. Group (3) and (4) were treated with P. granatum extract (mesocarp part) (250 mg/kg and 500 mg/kg body weight in water; respectively). Group (5) and (6) were treated with P. granatum extract (epicarp part) (250 mg/kg and 500 mg/kg body weight in water; respectively). Groups (7), (8) and (9) were treated with synthetic drugs Ranitidine (50 mg/kg body weight in water), Omeprazole (20 mg/kg body weight in water) and Sucralfate (360 mg/kg body weight suspended in 2% gum acacia) ]21[; respectively. Sample collection At the end of experiments, animals were subjected to light ether anesthesia, blood samples were withdrawn by retro-orbital bleeding and collected in EDTA-centrifuged tube (BD Biosciences, CA, USA). The plasma were separated by centrifugation at 2000 rpm for 15 min at 4°C, aliquotted, and stored at -80°C for further investigations. All rats were sacrificed after plasma collection. Gastrectomy specimens were removed from the control, ulcerated and treated groups, opened along the greater curvature, and thoroughly rinsed with normal saline, fixed and processed for haematoxylin and eosin (H&E) staining. Histopathological examination The gastrectomy specimens for each rat were fixed in 10% neutral buffered formalin solution. Each specimen was assessed grossly for dissection and detection of ulcer. Each specimen dehydrated in a graded alcohol series. After xylene treatment, the specimens were embedded in paraffin blocks. Five-micron thick

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ISSN: 0975-8585 sections were cut and stained with H&E staining and the mucosal and submucosal changes were examined by light microscopy. Damaging effect of ethanol-induced ulcer in both mucosa and submucosa; ulceration, erosion, congestion, inflammatory infiltrates type and intensity, epitheliotropism, edema, and reactive atypia were investigated in addition to reparative changes; angiogenesis and restoration of epithelium. Unintentional bias was prevented by coding rat's tissue samples. Immunohistochemical (IHC) analysis of COX-2 IHC staining was performed on formalin fixed, paraffin embedded material that were sectioned at 5 μm thickness and placed onto positive charged slides. COX-2 mouse monoclonal antibody (Santa Cruz Biotechnology, INC., U.S.A.) (200 μg/ml) was diluted 1:100 by phosphate buffered saline (PBS). IHC staining was performed using the labeled streptavidin biotin (LSAB)+System-HRP (Dakocytomation, Glostrup Denmark). All slides were de-paraffinized using xylene then dehydrated in decreasing concentrations of ethanol. After inhibition of endogenous peroxidase activity using 0.3% hydrogen peroxidase for 15 min, antigen retrieval using microwave heating (20 min; 10 mM Citrate buffer, pH 6.0) was performed. The slides were incubated overnight with anti-COX-2 primary antibody at room temperature, and washed by PBS then incubated with biotinylated secondary antibody for 15 min. After washing with PBS, the detection of bound antibody was accomplished using a modified labeled avidin–biotin reagent for 20 min. A 0.1% diaminobenzidine (DAB) (Sigma Chemical Company, St. Louis, MO) was used for 5 min as a chromogen. Slides were counterstained with Mayer’s hematoxylin for 5–10 min. Measurement of plasma angiogenesis markers [vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF)] Levels of VEGF and PDGF were quantified by enzyme linked immunosorbent assay (ELISA) as previously described by Talaat et al. ]22[. Absorbencies were measured at 450nm using ELISA plate reader (Sunrise, Tecan Group Ltd.). The ELISA reader-controlling software (Softmax) processes the digital data of raw absorbance value into a standard curve from which cytokine concentrations of unknown samples can be derived directly. Results were expressed as pictogram of cytokine per milliliter (pg/ml). Measurement of pro-inflammatory cytokines [interleukin -1 (IL-1β), IL-6 and Tumor necrosis factor-alpha (TNF-α)] Total concentrations of IL-1β, IL-6 and TNF- in plasma samples were measured using a commercial ELISA kit (RayBiotech, Inc. Norcross GA) according to the manufacturer’s instructions. Statistical analysis All statistical analyses were performed using Statistical Package for Social Science (SPSS) version 13 (SPSS, Inc., Chicago). Data are presented as means with corresponding SE. Comparisons between different groups were performed by one-way analysis of variance (ANOVA). Tukey’s test was used as a post-hoc test. Pathology data was calculated using MedCalc statistical software for biomedical research. Correlation among variables was determined using Pearson’s correlation test. A P value of p 0.05,**P>*Significance between treated groups and ulcerated-untreated group. (Meso, Mesocarp; Epi, Epicarp). ).*P 0.001.

Epi-Ex 500 and Ranitidine displayed near similar healing process indicators as regarding to the histopathological parameters with statistically insignificant differences between them. On the other hand, EpiEx 500 had a statistically significant ability to enhance healing and reducing the damaging effect as compared with Omeprazole and Sucralfate (p